Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels.

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Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26. Published in final edited form as: Curr Mol Pharmacol. 2015 ; 8(2): 188–205.

Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels Trainees of the Calcium Signals Laboratory and David T. Yue* Calcium Signals Laboratory, Departments of Biomedical Engineering and Neuroscience, Center for Cell Dynamics, Johns Hopkins University School of Medicine

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Abstract Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though longsought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

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Keywords Sodium Channels; Calcium Channels; Calmodulation

Introduction

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In recent years, the small 17 kDa bilobal Ca2+-binding protein calmodulin (CaM) has emerged as a potent and pervasive modulator of ion channels, often serving as an obligatory channel subunit [1]. The tale of CaM interaction with ion channels is entwined with rich mechanistic insights, important biological ramifications, and promises of crucial therapeutic insights. In this context, an early result that highlighted the role of CaM in regulating ion channel function came about from the genetic dissection of motile behavior in Paramecium. Strikingly, mutations in the CaM gene resulted in organisms that were either ‘underexcitable’ or ‘over-excitable’ to certain stimuli, due to the selective loss of either a Ca2+dependent Na current or a Ca2+-dependent K current [2]. This functional bipartition of CaM regulation foreshadowed a major theme of ion channel regulation, where the two lobes of

Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, voice: (410) 955-0079, fax: (410) 955-0549, Please direct correspondence to Manu Ben-Johny at [email protected]. *Dr. David T. Yue passed away on Dec. 23 2014. He was a remarkable scientist, mentor and friend. The world will miss this luminary scientist.

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CaM impart distinct functions. Since then, the catalogue of ion channels identified to be under the charm of CaM has been ever-expanding [3], and the list now includes diverse channel families such as the small-conductance potassium (SK) channels [4], the KCNQ potassium channels [5–7], cyclic nucleotide-gated channels [8], N-methyl D-aspartate (NMDA) receptor [9, 10], transient receptor potential (TRP) channels [11], ryanodine receptors [12], voltage-gated Ca2+ channels [13–16], and voltage-gated Na channels [17]. Amongst these channel families, CaM regulation (calmodulation) of voltage-gated Ca2+ and Na channels is a prominent prototype for ion channel modulation with diverse functional effects, exquisite Ca2+ decoding capabilities, and profound biological implications for cellular excitability. Here, we review recent advancements in our understanding of calmodulation of Ca2+ and Na channels – two fields that have long-shared a parallel history that now converges with exciting synergy.

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The rise of Ca2+ channel calmodulation Calmodulin regulation of Ca2+ channels is a prominent feedback mechanism that tunes channel gating to adjust vital Ca2+ entry in accordance with cytosolic Ca2+ signals. The first signs of this modulation emerged in 1978 when Brehm and Eckert observed that increased cytosolic Ca2+ could accelerate the inactivation of Ca2+ currents in Paramecium [18]. Thus, unlike the canonical voltage-dependent inactivation (‘h-gate’) of Hodgkin and Huxley [19], inactivation of Ca2+ currents was highly sensitive to cytosolic Ca2+ concentrations [20]. Indeed, this modulation allows the cell to sculpt electrical activity based on the integration of cytosolic spatiotemporal Ca2+ dynamics.

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CaV1 (L-type) channels serve as a prominent example of the regulatory principles and the molecular underpinnings of calmodulation. For many L-type channels, calmodulation manifests as a rapid Ca2+-dependent inactivation (CDI) evolving typically over 50 – 100 milliseconds. A convenient experimental approach to characterize CDI with quantitative precision is illustrated in Figure 1A. Here, a step depolarization in membrane potential activates Ca2+ channels, furnishing Ca2+ entry that triggers CDI mediated by CaM. By contrast, calmodulation is absent when Ba2+ is the permeant ion through the channel, thus serving as a negative control. Figure 1B displays the typical electrophysiological profile of Ca2+-dependent inactivation (CDI) as exemplified by CaV1.3, an archetypal channel with strong and unambiguous Ca2+/CaM regulation. In this manner, the steady-state decrement in Ca2+ current (red) compared to Ba2+ (black) serves as a robust quantitative metric for Ca2+ regulation of L-type Ca2+ channels, enabling the systematic characterization of underlying biophysical mechanisms and their biological implications [21, 22].

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At the single molecule level, Ca2+ dependent inactivation switches Ca2+ channels from a high open-probability (PO) mode of gating (‘mode 1’) to a lower PO gating mode (‘mode Ca’) in a quantized manner [23]. In ‘mode Ca’, channel openings are more sparse with a prolonged first latency [23]. The transition of channels from ‘mode 1’ to ‘mode Ca’ is governed by Ca2+/CaM. This behavior is notably different from the canonical voltagedependent inactivation of ion channels, where channels enter an absorbing non-conductive inactive state [24]. Indeed, systematic mapping of the state-dependence of calmodulation suggests that entry into ‘mode Ca’ proceeds equally from open and nearby closed states [21,

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25]. By contrast voltage-dependent inactivation of Ca2+ channels appears to proceed preferentially from the open state [25]. Importantly, as Ca2+ influx through Ca2+ channels drives calmodulation, shifts in voltage-dependence of channel activation could indirectly alter the strength of channel regulation [21]. Of pathophysiological relevance, various channelopathic mutations that result in heritable disorders including Timothy syndrome [26, 27], Autism Spectral Disorders [28], and Familial Hemiplegic Migraine [29, 30] cause shifts in channel activation. These indirect effects of channel gating on CaM regulation are important considerations in the effort to develop effective therapeutic strategies.

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The physiological phenomenon of Ca2+ regulation of L-type Ca2+ channels was wellestablished in many native systems including the heart, long before the definitive role of CaM in this process was confirmed [31–33]. We first consider key findings that led to the establishment of CaM as the Ca2+ sensor for Ca2+ channel regulation. The earliest proposals for the mechanism of Ca2+ channel CDI hypothesized both the direct binding of Ca2+ ions to the channel [34, 35] and Ca2+-dependent phosphorylation and/or dephosphorylation of channels [36, 37]. Even then, the direct or indirect role of CaM in this regulatory process was suspected, but remained unclear for many years [20, 37]. The molecular cloning and functional expression of recombinant Ca2+ channels in heterologous systems, paved the way towards an in depth structure-function understanding of Ca2+ regulation [38]. The Ca2+ channel is a large protein complex composed of roughly 2000 amino acids in the main αsubunit. Identifying a molecular segment relevant for Ca2+ regulation on the channel complex thus seemed daunting. Some hope was garnered following the identification of a putative Ca2+-binding ‘EF hand’ motif on the carboxy-terminus of both Ca2+ and Na channels through bioinformatic analysis (Figure 1C, EF) [39]. Classically, the EF hand motif is composed of a helix-loop-helix protein segment with acidic residues in the loop region spatially arranged to coordinate Ca2+ ions [40]. The channel EF hand, however, appeared to be vestigial in that it lacked key acidic residues essential for Ca2+ binding [39]. Further analysis of the channel carboxy-terminus revealed a second EF-hand motif analog [41, 42]. The field quickly focused on this channel locus and evaluated its role in CDI. To this end, it was fortuitous that the L-type (CaV1.2) channels exhibited strong CDI under elevated intracellular Ca2+ buffering, while the R-type (CaV2.3) channels demonstrated only weak CDI under the same conditions rendering them useful for chimeric channel analysis [43, 44]. Accordingly, segments of the carboxy-tail (Ca2+-inactivating region; CI region) of CaV1.2 and CaV2.3 channels were swapped and Ca2+ regulation was quantified [44]. The proximal third of the channel carboxy terminus and, more specifically, the dual vestigial EF hand segment of CaV1.2 emerged as critical segments for strong Ca2+ regulation. It was then plausible that Ca2+ may directly interact with the channel to trigger CDI as postulated earlier [20, 34, 35]. However, point mutations of key residues in the Ca2+ binding loop of the EF1 segment spared CDI, arguing that Ca2+ does not directly interact with the channel itself [45, 46]. Overall, it was clear that the Ca2+ channel CI region, including the two vestigial EF hand regions, played a prominent role in calmodulation, though the precise function of these segments remained poorly defined. Cognizant of these results, the search-space for a structural determinant of Ca2+ channel Ca2+ regulation soon expanded to include alternative segments of the channel CI region. Systematic deletions of overlapping segments of the CaV1.2 channel CI region identified an Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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IQ motif (Figure 1C) as yet another critical element in the CDI process [47]. The IQ domain, named for conserved isoleucine and glutamine residues, is a well-known target of Ca2+independent CaM binding [48]. Moreover, mutagenesis of the CaV1.2 IQ domain confirmed a critical role for this segment in CDI and renewed interest in the possibility that CaM could still be the Ca2+ sensor for Ca2+ channels [47, 49]. That said, prior experiments utilizing pharmacological inhibitors of CaM had failed to eliminate CDI of L-type Ca2+ channels [23, 47]. The resolution to this conundrum came from the development of a Ca2+-insensitive mutant CaM, where point mutations were introduced within all four EF-hand Ca2+ binding sites (CaM1234) [50]. If CaM were to associate with the Ca2+ channel complex in a Ca2+ independent manner, then this mutant CaM could act as a dominant negative to eliminate regulatory function. Indeed, CaM was identified as the Ca2+ sensor for the SK channels using such a maneuver [50]. Accordingly, when CaM1234 was coexpressed with CaV1.2 channels, CDI was eliminated, establishing CaM as the unambiguous Ca2+ sensor and gave birth to the concept of ‘calmodulation’ of Ca2+ channels [51, 52]. Importantly, Ca2+-free CaM (apoCaM) was subsequently shown to bind the CaV1.2 CI region with a strong dependence upon the IQ domain [53–56]. Thus, CaM acts as a resident Ca2+ sensor molecule in the Ca2+ channel signaling complex. Since this discovery, nearly all Ca2+ channels have been shown to be regulated by CaM with diverse functional caricatures and elegant Ca2+ sensing properties [43, 57–63], as described in subsequent sections. More generally, the usage of a dominant negative CaM (CaM1234) remains an indispensable tool for establishing CaM as a Ca2+-sensor for various macromolecular targets where preassociation of apoCaM is a requisite.

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The mechanism of CaM action on the channel and the functionally relevant CaM/channel interfaces has continued to be controversial. A prominent hypothesis has been an ‘IQcentric’ model based on atomic structures of various Ca2+ channels in complex with Ca2+/CaM [16, 64–67]. The merits and weaknesses of this model have been reviewed at length [15, 16]. Briefly, mutations in the IQ domain markedly alter Ca2+ regulation of many Ca2+ channels [41, 43, 47, 49, 52, 54, 61, 66–71]. ApoCaM preassociation has been shown to require the IQ domain [41, 53–55, 69] and Ca2+/CaM also binds to the IQ domain as a peptide with high affinity [41, 43, 51, 52, 55, 56, 69, 70, 72, 73]. However, atomic structures of Ca2+/CaM bound to wild-type and mutant IQ peptides of CaV1.2 show that the key isoleucine residue is deeply buried [64, 74], and that alanine substitution of this residue does not significantly alter CaM binding conformation [64]. It is unclear how a subtle change at this locus entombed within the hydrophobic Ca2+/CaM interface could alter calmodulation [16, 64]. Further, several mutations in the channel IQ domain that disrupt CDI do not alter the binding affinity of Ca2+/CaM for IQ peptides [41, 69]. As well, mutations that do weaken Ca2+/CaM binding to IQ peptides do not alter the strength of CDI [41, 69]. Together, these results argue against a simple ‘IQ centric’ model leading to the possibility that alternative CaM interacting sites on the Ca2+ channel may bear critical functional relevance. In part, the complexity in understanding the mutational effects on Ca2+ channel CDI stems from an incomplete consideration of the whole CaM regulatory system [41, 69]. In Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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particular, many studies have ascribed functional deficits in CDI resulting from mutations within Ca2+ channels to be a disruption of the Ca2+/CaM interface on the channel. However, as calmodulation entails a system of configurational changes, mutations on the channel may affect the propensity for any number of channel states. A general conceptual framework for understanding the various states that underlie calmodulation is illustrated in Figure 1D [41]. In configuration E, channels lack apoCaM preassociation. As such, these channels may not undergo calmodulation [41, 58, 59, 69, 75]. Recently, this configuration has also been identified to represent a low PO gating mode [76]. Upon apoCaM binding, channels traverse into configuration A. In this state, channel openings occur normally and the channels are primed to undergo calmodulation [41, 69]. As the two lobes of CaM often act semiindependently, we may further delineate the CaM regulatory process into multiple steps. When Ca2+ binds to the C-lobe of CaM, this lobe may interact with an appropriate channel interface yielding configuration IC with a reduced PO. Alternatively, once calcified, the Nlobe Ca2+/CaM may interact with its effector site to yield configuration IN, which also has a reduced PO. Finally, Ca2+ binding to both lobes of CaM yields configuration ICN, a fully inactivated channel with reduced PO. Importantly, the entry into configuration ICN may involve a positively cooperative step defined by a cooperativity-coefficient λ>>1.

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Thus formulated, mutations of channel interfaces may alter either a single transition or multiple transitions in this regulatory scheme. Nonetheless, the effects on individual transitions may be evaluated using either wild-type or mutant CaM. For example, if a mutation were to alter the binding interaction of the C-lobe of Ca2+/CaM to its effector interface, then the coexpression of mutant CaM12 (mutant CaM with Ca2+ binding enabled only to C-lobe) would isolate this component of CDI. Conversely, the N-lobe component of CDI could be isolated using CaM34 (a mutant CaM with Ca2+ binding enabled only to the N-lobe). In both cases, confounding effects of positive cooperativity are carefully avoided. If a mutation were to diminish apoCaM binding, then a fraction of channels may lack a preassociated apoCaM at baseline CaM levels in a cell. Under this condition, channels would preferentially reside in configuration E, incapable of undergoing calmodulation. However, overexpression of wild-type CaM would repopulate the channels with an apoCaM, thus redistributing them into configuration A by mass action. This maneuver would allow one to rescue a deficit in CDI resulting from a loss of apoCaM preassociation. This general framework allows one to dissect individual sub-configurations of calmodulation, thereby enabling the systematic characterization of relevant CaM/channel interfaces.

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So far, the general framework presented here assumes a single CaM elicits CaM regulatory function on the channel. However, the stoichiometry of CaM to the Ca2+ channel complex remains controversial [77–81]. Recent atomic structures of Ca2+/CaM complexed with subsegments of the CaV1.2 CI region suggest that multiple CaM molecules may be necessary for the full breadth of Ca2+ regulatory functions [77, 78]. Additionally, multiple peptides derived from Ca2+ channel cytosolic segments have been shown to interact with Ca2+/CaM [73, 82–85]. From the perspective of channel function, the stoichiometry of the preassociated apoCaM may be more relevant, as the exchange of Ca2+/CaM from the cytosol to the channel alcove is unlikely given the brevity of voltage-pulses used to probe Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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calmodulation (Figure 1A–1B) and the relative abundance of Ca2+/CaM targets in the cell [86]. Live-cell FRET (Fluorescence Resonance Energy Transfer) binding experiments point to a 1:1 stoichiometry of apoCaM binding to the holo-CaV1.2 channel complex [81]. Functional experiments have also shown that the covalent fusion of a single CaM molecule to CaV1.2 and CaV1.3 channels is sufficient to elicit CDI [79, 80]. Overall, the 1:1 stoichiometry of CaM with Ca2+ channel complex simplifies the configurational states necessary to understand calmodulation, thus avoiding the complexity observed in systems that utilize multiple CaM molecules [87]. A possible implication of the singular stoichiometry is that the lobes of CaM may bridge various closely-juxtaposed Ca2+/CaM binding sites on the channel. For example, a recent biochemical study highlights the preference of an amino-terminal Ca2+/CaM binding segment (NSCaTE, N-terminal Spatial Ca2+ Transforming Element) to interact with a single lobe of CaM (N-lobe) [83, 88]. Furthermore, this NSCaTE element can interact with Ca2+/CaM prebound to an IQ domain peptide, suggesting the possible bridging of the channel amino- and carboxy-termini [89].

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Thus apprised, recent studies have formulated a new structure-function framework of CaM/ channel configurations that underlie calmodulation of Ca2+ channels as illustrated by molecular models in Figure 1E–1F for CaV1.3 channels [41, 69]. This molecular framework was built based on functional characterization of extensive alanine scanning mutagenesis of the CaV1.3 CI region, and the subsequent correlation of functional deficits in calmodulation to underlying binding interactions [41, 69]. First, a single apoCaM preassociates to the Ca2+ channel carboxy terminus with the C-lobe of apoCaM bound to the channel IQ domain, and the N-lobe bound to the channel dual vestigial EF-hand segments [41, 54, 55, 69]. The ab initio structural model of the CaV1.3 carboxy-terminus in Figure 1E shows two vestigial EF hand segments (EF, green), a protruded helix representing the preIQ domain (green), and a helical IQ domain (blue). The structural models of the preIQ segment and the IQ domain correspond to known atomic structures of CaV1.2 channel peptides [64, 65, 77, 78]. Overall, the new consensus is that apoCaM preassociates with the Ca2+ channel CI region through a bipartite binding scheme that enhances CaM affinity through positive cooperativity.

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Upon Ca2+ binding to CaM, Ca2+/CaM is proposed to rearrange itself on the channel complex to elicit configuration ICN (Figure 1F). In this model, the Ca2+-bound N-lobe associates with the NSCaTE module on the channel amino terminus to elicit N-lobe CDI, as illustrated by the NMR structure depicted in Figure 1F [88]. The Ca2+ bound C-lobe of CaM has been argued to form a tripartite complex with the channel dual vestigial EF-hands and the IQ domain (Figure 1F) [41]. This ternary arrangement explains the importance of both the IQ domain and the dual vestigial EF-hand segments as determined in early characterization of Ca2+ channel CDI [44, 47, 51]. Interestingly, the C-lobe configuration likely corresponds to a traditional CaM/peptide complex, where the channel dual vestigial EF hands may serve as a surrogate lobe of CaM. Lastly, apoCaM itself has been found to be a potent modulator of channel PO [76]. In the past, it was believed that apoCaM affinity was sufficiently high that all channels must possess a preassociated CaM [53, 55]. The discovery of alternative splice variants of CaV1.3 [90, 91] and CaV1.4 channels [92] demonstrated the presence of a distal carboxy-terminal autoinhibitory domain (ICDI segment) that could compete with apoCaM, thus refining this


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perspective [58, 59, 75, 93, 94]. The CaV1.3 channel IQ domain has also been found to be post-transcriptionally altered through RNA editing [95]. All these post-transcriptional modifications alter channel function by essentially weakening apoCaM preassociation [58, 59, 69, 75, 93]. As such, these CaV1.3 channel variants support variable strengths of CDI [59, 69, 90, 95, 96]. These channel variants have also been observed to possess a substantially diminished baseline PO [76, 96]. However, it was not initially clear whether this enormous diversity in CDI and baseline PO of CaV1.3 channel variants related to one and the same mechanistic process. Reassuringly, the overexpression of wild-type CaM rescued both CDI and baseline PO of channel variants with weakened apoCaM preassociation [69, 76]. Accordingly, a simple scheme that unifies the mechanistic underpinnings of these diverse channel variants emerges – apoCaM binding itself can enhance the baseline PO of L-type Ca2+ channels by nearly 7-fold and also prime them for calmodulation. Much like the switching of channels from ‘mode 1’ to ‘mode Ca’ during calmodulation, single-channel records of these channel variants depicted quantized behavior whereby channels switched from a high PO gating mode to a low PO gating mode [76]. Indeed, transient elevation of CaM near the Ca2+ channels using novel chemical biological tools strongly enhanced peak Ca2+ currents in channel variants lacking an apoCaM. The unified conceptual scheme is as follows – apoCaM itself enhances channel PO, and Ca2+binding to CaM relieves this baseline enhancement [76]. The biological implications of these mechanisms are far-reaching. Of note, apoCaM affinities of many CaV1.3 channel variants are such that natural fluctuations of ambient CaM in native cells could redistribute channels between pools that lack CaM and ones charged with apoCaM [69, 75, 76]. This switch in channel distribution could profoundly alter both the maximal Ca2+ current as well as the shape of the Ca2+ waveform in response to electrical stimulation of a cell.

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Overall, the proposed structure-function framework in Figure 1E–1F reveals important channel interfaces with implications for the rational design of drugs that could alter Ca2+ channel function. For example, molecules that target apoCaM/CI region interface could serve as novel inhibitors of Ca2+ channels. Additionally, the switching of CaM/channel interactions upon Ca2+ binding implies drugs that target CaM effector interfaces could allosterically abolish Ca2+ channel inactivation without altering the peak current. In fact, natural molecules like CaBP (Ca2+-binding protein) may exploit this mechanistic principle to switch off calmodulation [97]. Lastly, this framework will help guide future structural studies and is likely to serve as a model for understanding calmodulation of other channels including voltage-gated Na channels.

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Calmodulation underlies a pervasive mechanism across the entire CaV1/2 channel family. Beyond the tuning of channel function by apoCaM, Ca2+/CaM regulation is capable of multiple modes of Ca2+ decoding and channel modulation. To a large extent, this customization of channel regulation is determined by the binding of Ca2+ to either the C- or N-lobe of CaM such that each lobe is capable of evoking distinct channel modulations [43, 61, 62, 70, 98–100] that allow for exquisite spatial and temporal decoding of Ca2+ signals. For example, CaV2.1 channels demonstrate functional bipartitioning of CaM in channel regulation. For these channels, Ca2+ regulation manifests as a rapid Ca2+-dependent Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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facilitation (CDF) of Ca2+ current [101], followed by a slowly developing CDI. This dual regulation can be clearly observed in the response of Ca2+ currents to a train of action potentials (Figure 2A; CDF, circle; CDI, square). Moreover, these disparate regulatory effects can be triggered independently by the two lobes of CaM [70, 71]. Specifically, the Clobe of CaM triggers CDF, whereas the N-lobe is responsible for CDI [70, 71]. Thus, each lobe of CaM can mediate signaling to a separate set of functions.

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While the channel structural determinants of CDI versus CDF remain unknown, the dual regulation of Ca2+ channels by each lobe of CaM generalizes across channel subtypes. In most CaV1 channels, the C-lobe induces a kinetically distinct rapid phase of CDI, whereas the N-lobe yields a slower component [51, 61, 83]. In some studies, CDF has also been described in CaV1.2 channels, but the underlying mechanism remains mysterious. In Ca2+ channels of native cardiomyocytes, there is a weak manifestation of CDF which can be attenuated by blocking Ca2+ release from neighboring ryanodine receptors [102]. In addition, CDF of CaV1.2 channels in recombinant systems can only be viewed in the presence of channels bearing point mutations within the IQ domain, and only when expressed in frog oocytes [68]. Curiously, CDF is not observed in recombinant CaV1.2 channels expressed in mammalian cell lines [51]. By contrast, CDI of CaV1.2 channels is strong and universally observed across experimental platforms. Nonetheless, this latent CDF may provide shared mechanistic insight for Na channel regulation as described later.

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Beyond simply splitting the Ca2+ signal, Ca2+/CaM-dependent feedback exhibits remarkable spatial selectivity which can be revealed by probing channels in the presence of variable Ca2+ buffering. Some types of calmodulation are insensitive to strong intracellular Ca2+ buffering (e.g., CaV1.2 CDI), whereas others are not (CaV2.3 CDI). Strong Ca2+ buffering would spare the Ca2+ signals near the cytoplasmic face of channels where the large influx of Ca2+ would readily overwhelm the available buffers (Figure 2B, local Ca2+ signal) [103, 104]. This local signal is sufficient to evoke C-lobe CaM regulation. And the local Ca2+ selectivity allows for privileged signaling within a single CaM/channel complex without interference from neighboring Ca2+ sources. By contrast, the N-lobe of CaM generally imparts a regulatory effect only in the presence of low Ca2+ buffering, a condition that permits global elevation of cytosolic Ca2+ (Figure 2B, global Ca2+ signal). Thus, the N-lobe of CaM is able to ignore the intense local Ca2+ fluctuations and respond to the smaller global elevation in Ca2+ concentration [43, 70, 105, 106]. Such global selectivity of the Nlobe of CaM enables a resident Ca2+ sensor molecule to respond to remote Ca2+ signals, thus coordinating Ca2+ regulation across large portions of a cell [99, 100].

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Further evidence of the local versus global selectivity of each lobe of CaM comes from single channel recordings of CaV2.1. In this mode, only a single channel is activated and therefore only a local Ca2+ signal exists. Probed thus, CaV2.1 channels exhibit CDF (driven by C-lobe), but not CDI (triggered by N-lobe) [105]. Similar results have also been shown for CaV1.2 channels [23]. These results confirm the proposed differential selectivity of each lobe of CaM. By 2006, a general consensus appeared to converge for CaV1/2 channels. The N-lobe of CaM inevitably acted as a global Ca2+ sensor, while the C-lobe functioned as a local sensor.


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This perspective was revised upon closer examination of CaV1.2/1.3 CDI [83]. These channels exhibited both N and C-lobe CDI even in the presence of strong intracellular Ca2+ buffering. This divergence in N-lobe selectivity is due to the NSCaTE channel segment conserved on the amino-terminus of CaV1.2/1.3 channels. NSCaTE is a Ca2+/CaM binding site whose presence augments the overall affinity of the channel for Ca2+/CaM over apoCaM [82, 83]. The existence of this element endows the N-lobe of CaM with local Ca2+ sensitivity in these channels. Elimination of the NSCaTE site then switches their N-lobe CDI to a global profile. Likewise, donation of the NSCaTE segment to CaV2 channels results in N-lobe CDI with local Ca2+ selectivity [83]. In this manner, the presence or absence of the NSCaTE module within the amino-terminus of a channel can tune the spatial Ca2+ selectivity of N-lobe mediated channel regulation.

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The modularity of the NSCaTE element revealed that the local/global selectivity of each lobe of CaM is not immutable. The contrasting spatial Ca2+ selectivities can be explained as emergent properties of a system of channel transitions where a lobe of apoCaM transiently unbinds from a preassociation site before binding Ca2+ ions, and then reengages an effector interface upon Ca2+ binding to elicit channel regulation. The slow Ca2+ unbinding kinetics of the C-lobe of CaM invariably confers local Ca2+ selectivity. The rapid Ca2+ unbinding kinetics of the N-lobe of CaM, on the other hand, renders this lobe a global Ca2+ sensor. However, as the channel affinity for Ca2+/CaM is enhanced, the system shifts to favor local Ca2+ sensitivity [107]. Thus, the N-lobe of CaM is a tunable Ca2+ sensor [107]. The full scheme of CaM regulatory function for Ca2+ channels is illustrated in Figure 2C. These remarkable Ca2+ decoding principles may be a general scheme that confers distinct spatial Ca2+ selectivities to a wide-range of biological molecules.

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The rise of Na channel calmodulation

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Developments in the understanding of Ca2+ regulation of Na channels closely paralleled the discoveries of Ca2+ channel calmodulation, though the functional outcome and underlying modulatory principles often appeared to diverge (Figure 3A). The first postulate that intracellular Ca2+ ions might regulate Na channels emerged with the early bioinformatic analysis that demonstrated the presence of a conserved vestigial EF hand segment in the carboxy-terminus of both Na and Ca2+ channels [39]. However, this possibility was nearly forgotten for almost a decade before pioneering biochemical studies explicitly demonstrated the robust binding of both apoCaM and Ca2+/CaM to the Na channel IQ domain peptides [108]. This study also identified a second closely juxtaposed Ca2+/CaM binding site, termed ‘Baa motif,’ immediately downstream of the Na channel IQ domain [108]. While these biochemical experiments demonstrated CaM interaction with Na channels, no functional outcome for this binding was yet ascribed [108]. A prominent feature of voltage-gated Na channels is its voltage-dependent inactivation [19, 109]. As such, early studies hypothesized that CaM and/or the channel vestigial EF hand segment might modulate particular attributes of Na channel voltage-dependent inactivation [110, 111]. Fitting with this idea, the Na channel carboxy-terminus was shown to alter fast inactivation properties through chimeric channel analysis of the neuronal NaV1.2 and cardiac NaV1.5 channels [112]. However, the effects of Ca2+ on Na channels often remained


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controversial and isoform specific as reviewed in the past [65]. Typically, these effects were probed by characterizing Na current properties in two populations of cells dialyzed with either low or elevated Ca2+ (Figure 3B). For the cardiac NaV1.5 channels, intracellular Ca2+ was shown to induce a subtle ~10 mV depolarizing or right shift in the voltage-dependence of steady-state inactivation or h∞ curves (Figure 3C) – an effect that would contribute to an enhancement in Na channel availability during periods of high activity [42, 110, 113, 114]. However, this modulatory effect was not universally accepted with other studies reporting no significant effect of Ca2+ on NaV1.5 steady-state inactivation curves [111, 115, 116]. For skeletal muscle NaV1.4 channels, an initial report suggested a subtle Ca2+-induced hyperpolarizing or left shift in voltage-dependence of steady-state inactivation curves [111], although later studies reported no significant shift of NaV1.4 steady-state inactivation curves [115, 117]. The wild-type neuronal NaV1.2 channel was reported to be insensitive to Ca2+, however a channelopathic mutation within the IQ domain associated with autism (R1902C) was found to unveil a hyperpolarizing or left-shift in steady-state inactivation [118]. By contrast, the wild-type neuronal NaV1.1 channels were argued to possess a subtle hyperpolarizing shift in voltage-dependence of steady-state inactivation in response to Ca2+ [119]. The neuronal NaV1.6 channels showed no significant shift in steady-state inactivation curves [117]. Overall, the full spectrum of modulatory effects of Ca2+ on Na channels appeared to be complex and idiosyncratic, evading consensus for nearly a decade.


The molecular mechanisms underlying these variegated functional effects of Ca2+ on Na channels were often contested. Of note, the identity of the Ca2+ sensor for Na channel regulation was debated [65], reflecting similar controversies in the Ca2+ channel field [16]. One possibility was the direct binding of Ca2+ ions to the channel dual vestigial EF hand segment. Early studies proposed putative Ca2+ coordinating residues in this segment based on sequence alignment [42]. Alanine substitution of these proposed critical acidic residues (termed ‘4X’, corresponding to E1788, D1790, D1792, and E1799 in the human NaV1.5 channel) abolished Ca2+-sensitive shifts in voltage-dependence of steady-state inactivation observed for NaV1.5 channels [42, 120]. In this model, the IQ domain was suggested to alter Ca2+ sensitivity of the channel dual vestigial EF hand segment allosterically [121]. Fitting with this idea, the Na channel IQ domain has been argued to interact with the dual vestigial EF motifs also on the carboxy-terminus of the channel [121, 122]. Nonetheless, later structural studies demonstrated that these proposed critical residues (‘4X’) were arranged spatially in an orientation incompatible with Ca2+ coordination [118, 123–126]. Moreover, even in the presence of high Ca2+ concentrations, the atomic structures of both NaV1.5 and NaV1.2 failed to exhibit Ca2+ binding to the channel dual vestigial EF hand [118], suggesting that the functional effects observed with the ‘4X’ mutant may have been the result of indirect mechanisms. The role of CaM in Ca2+ regulation of Na channels has been controversial with some studies proposing a prominent Ca2+ sensing role [115, 118, 127, 128], while others arguing for subtle secondary functions [120, 121, 125]. The direct role of CaM was envisioned based on biochemical studies that suggested strong interaction between the channel IQ domain and both apoCaM and Ca2+/CaM [108]. Biochemically, CaM was shown to support a configurational change of the NaV1.2 carboxy terminus in the context of a channelopathic mutation [127]. Functionally, some role for CaM in Ca2+ regulation of NaV1.5 channels was Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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proposed based on the finding that Ca2+/CaM could interact with the intracellular linker between domain III and domain IV (III-IV loop) of the NaV1.5 channel [114, 128, 129]. In terms of overall Na channel function, the III-IV loop has been argued to serve a privileged role in eliciting the canonical fast-inactivation [130–132]. As such, it is conceivable that Ca2+/CaM interaction with the Na channel III-IV loop might modulate fast inactivation. In this model, the C-lobe of CaM was proposed to migrate from the channel IQ domain to the III-IV loop upon calcification [114, 128]. In other studies, CaM was thought to play an indirect role in controlling channel steady-state inactivation that is revealed only upon disabling the dual vestigial EF hand Ca2+ sensing machinery [120].


Finally, a few methodological nuances utilized in these experiments merit attention. First, nearly all studies employed either EGTA (ethylene glycol tetraacetic acid) or BAPTA (1,2bis(0-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) to buffer Ca2+ in the 0.5 – 10 µM range, a concentration well above their dissociation constant (Kd ~ 100 nM) [133]. As such, contaminant Ca2+ ions may lead to drastic fluctuations in free Ca2+ concentration, rendering Ca2+ essentially unbuffered [115, 133]. Second, many of these studies also utilized F− anions in internal solutions to facilitate patch clamp experiments. However, F− ions have been shown to alter G-protein signaling status in cells[134], among other reported effects [65] and thus may regulate Na current through Ca2+-independent pathways [135]. Third, prior to measurement, time-dependent voltage shifts in steady-state inactivation curves are typically allowed to equilibrate following the onset of cell pipet dialysis [120]. Variability in the actual equilibration process could obscure the precise quantification of Ca2+ dependent effects on Na current. Another limitation of Ca2+ delivery via pipet dialysis concerns the difficulty of detecting fast Ca2+-induced changes in current amplitude without an overt change in the voltage-dependence of activation or inactivation of the channel. Changes in current amplitudes may relate to either disrupted channel gating or trafficking and could arise from numerous cellular factors. These challenges likely contributed to the conflicting reports, and point to the need for a robust and quantitative assay to probe Ca2+ effects of Na channels.

Rapid Ca2+ delivery reveals rapid Na channel modulation

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A key methodological advance is the usage of simultaneous patch-fluorimetry and rapid Ca2+-photouncaging. This approach had been used recently to quantitatively assess Ca2+regulation of CaV1.3 channels [136]. A schematic of this experimental method is illustrated in Figure 4A (left subpanel). Here, caged-Ca2+ molecules prepared from mixtures of a photolabile derivative of EDTA, DM-Nitrophen or 1-(2-Nitro-4,5-dimethoxyphenyl)-1,2diaminoethane-N,N,N′,N′-tetraacetic acid, and Ca2+ ions are dialyzed into a cell along with a known ratio of the Ca2+ sensitive dye Fluo-4FF (Kd ~ 9.8 µM) and the red dye Alexa-568. Na currents were evaluated in response to a train of voltage step-depolarizations while quantitative Ca2+ measurements were obtained simultaneously using fluorescence measurements. Step increases in Ca2+ concentrations could be triggered at will using a brief UV light-pulse (Figure 4A, right subpanel). In this manner, Ca2+-dependent alterations in Na


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current could be evaluated and quantified with millisecond precision – each cell as its own control [115].

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Surprisingly, when the cardiac Na channel, NaV1.5, was studied in this manner, no significant Ca2+-dependent effect was observed [115]. Figure 4B depicts this experimental outcome. Briefly, NaV1.5 currents were evoked using a train of step depolarizations to 0 mV. The peak Na currents prior to Ca2+ uncaging (Figure 4B, gray dots) remained steady during the voltage-pulse protocol indicating negligible run-down of Na current. Moreover, Ca2+ measurements prior to Ca2+ uncaging showed resting Ca2+ to be 10 µM, a range well within the Ca2+ requirement for regulation of Na currents [139].


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Mechanistically, Ca2+ regulation of NaV1.4 currents appeared to be largely independent of voltage [115]. This regulation resulted in a uniform scaling-down of steady-state inactivation as opposed to a simple shift its voltage-dependence, suggesting that CDI may not be reversed by hyperpolarization of the cell [115]. Single molecule experiments of NaV1.4 channels further established Ca2+ regulation of Na channels as a genuine molecular-level effect of Ca2+ on channel PO [115]. In terms of channel gating, there are two possible molecular interpretations for NaV1.4 CDI. First, Ca2+ might fractionally reduce channel availability independent of voltage. Thus, CDI might correspond to a silent state. Alternatively, Ca2+ might alter the transitions between closed and open states of NaV1.4 akin to an alternate low PO gating mode associated with Ca2+ channel calmodulation [23, 105]. These distinct possibilities will have to be resolved through single molecule measurements of NaV1.4 channel gating. In all, these results firmly established the Ca2+ regulation of NaV1.4 channels – a modulation with uncanny similarities to Ca2+ regulation of Ca2+ channels.

Convergence of Ca2+ and Na channel calmodulation Several shared modulatory principles between Ca2+ and Na channel calmodulation have emerged in recent years (Figure 5A) [76, 115, 126]. First, Ca2+ regulation of Na channels now presents as a rapid and dynamic process that tunes Na channels to the fluctuations of cytosolic Ca2+. Indeed, like Ca2+ channel calmodulation, Na channel regulation is attained in a matter of ~100 milliseconds and can be readily reversed as cytosolic Ca2+ wanes [115]. As with Ca2+ channels, the kinetics of Na channel calmodulation steeply depends upon the amplitude of cytosolic Ca2+ eliciting the modulation [115, 136].


Second, commonalities in the mechanism of Ca2+ sensing by both Na and Ca2+ channels have also been identified. A combination of structural [118, 123, 124, 126] and functional studies [115] now argue strongly that the channel dual vestigial EF hands are unlikely to bind Ca2+ ions and support functional manifestations of calmodulation. Functionally, alanine substitution of key acidic residues in the Ca2+ binding loop of the Na channel dual vestigial EF hands failed to alter the Ca2+ sensitivity of Ca2+ regulation for NaV1.4 channels probed using Ca2+-uncaging [115]. Parallel results have been observed for Ca2+ channel regulation as discussed previously [45, 46]. Nonetheless, it remains possible that the dual vestigial EF hands may support other critical roles for Na channel regulation, much as has been observed for Ca2+ channels [41, 45]. By contrast, growing structural and functional evidence points to a fundamental role for CaM as the Ca2+ sensor for Na channels [115, 118, 124, 126]. A key experimental gap preventing the establishment of CaM as the Ca2+ sensor of Na channels has been the difficulty in inhibiting Ca2+ regulatory effect upon disabling CaM function [65]. To this end, a greatly enabling tool that established CaM as the unequivocal Ca2+ sensor for Ca2+ channels has been the shunting of Ca2+ regulation in the presence of dominant negative CaM (CaM1234) [43, 49, 51, 52, 61, 62, 70]. Likewise, the coexpression of CaM1234 with NaV1.4 channels resulted in a complete disruption of Ca2+ regulation, thus establishing CaM as the Ca2+ sensor for these channels [115]. Moreover, the functional bipartition scheme of CaM function established for Ca2+ channels likely extends to CaM regulation of Na channels. Ca2+ binding to the N-lobe of CaM is both necessary and sufficient to recapitulate the full Ca2+ regulatory function of NaV1.4 channels [115]. Fitting Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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with this functional role, a recent atomic structure of NaV1.2 carboxy-tail in complex with Ca2+/CaM has shown that only the N-lobe of CaM binds to Ca2+ ions [118]. Moreover, Na channel III–IV loop has been argued to be a functionally-relevant conserved interface for Ca2+/CaM [114, 128]. Similarly, Ca2+/CaM has been shown to interact with the CaV1.3 channel III-IV loop [115]. However, the binding of Ca2+/CaM to the III-IV loop of both channel families now appears to be functionally irrelevant [115]. Instead, CaM interaction with the channel carboxy-terminus emerges as the primary structural determinant for both Na and Ca2+ channels [47, 49, 53, 55, 115, 118, 124, 126, 140, 141]. Furthermore, a striking similarity between NaV1.4 and CaV1.2 channel CaM regulation is the curious phenomenon whereby substitution of isoleucine-glutamine residues in the IQ domain to dual alanines (NaV1.4 IQ/AA and CaV1.2 IQ/AA) unveils latent Ca2+-dependent facilitation for both channel families [52, 115].


Another burgeoning principle of calmodulation of Ca2+ channels is the effect of apoCaM binding to potently regulate channel PO [76]. Remarkably, this principle appears to hold true for Na channels as well [76]. Like the CaV1.3 channels, the wild-type NaV1.4 channels bind apoCaM with high affinity [76, 81, 117]. Disruption of CaM binding to the NaV1.4 channels through mutation of the IQ domain (NaV1.4 IQ/AA) results in channels with a very low PO under conditions of low CaM. Overexpression of CaM fully rescues the PO of these mutant Na channels, raising the possibility that CaM could tune Na channel peak PO in a Ca2+independent manner [76]. Fitting with this resonance in channel function, a recent atomic structure of the NaV1.5 carboxy-tail region in complex with apoCaM alone showed that the C-lobe preassociated with the IQ domain, and the N-lobe bound to the dual vestigial EF hand segment in a ‘lock-washer’ configuration [126]. This configuration is very reminiscent of the consensus apoCaM binding to the Ca2+ channel CI region as identified in recent functional studies [41, 69]. The apparent likeness in Na and Ca2+ channel regulatory function may correspond to a deep structural homology. It is worth noting that an alternate structure of a ternary complex of NaV1.5 carboxy-tail bound to both apoCaM and fibroblast growth factor homologous factor (FHF) showed a subtly different apoCaM binding configuration (‘lollipop arrangement’) [124]. In this model, the FHF molecule is bound to the dual vestigial EF hand segment while the N-lobe of apoCaM is dislodged from the channel interface. Resolving these structural differences and possible functional implications remains an open question. Importantly, the full spectrum of biological implications of apoCaM binding to Na channels is yet to be fully explored and represents an exciting new avenue of research.

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From the perspective of conservation, it is intriguing that nearly all high-voltage gated Ca2+ channels possess highly similar CI regions and elaborate robust and recognizably similar forms of CaM regulation (Figure 5B). The low voltage-activated (T-type) Ca2+ channels lack the CI region [142, 143] and are also insensitive to direct regulation by Ca2+/CaM (Figure 5B) [144]. All members of the mammalian voltage-gated Na channel family also possess the CI region [65]. So far, the NaV1.4 channel has been shown to be modulated by Ca2+/CaM in a manner highly similar to Ca2+ channel calmodulation. Although rapid Ca2+ regulation appears to be absent in wild-type NaV1.5 channels, molecular manipulations of this channel could confer latent Ca2+ regulation [115]. It remains to be seen how the static shifts in the


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voltage-dependence of steady-state inactivation previously reported for this channel relate to Ca2+ channel calmodulation. Nonetheless, the extensive structural and functional similarity between calmodulation of Ca2+ and Na channels suggests that the CI region may be an evolutionarily conserved module [115, 126]. Sequence alignment of the CI region of both Ca2+ and Na channels demonstrates striking conservation across major eukaryotic phyla [115]. Notably, the CI region can be evolutionarily traced to the single cell organism Paramecium, which only possesses a voltage-gated Ca2+ channel but not a voltage-gated Na channel [115]. It was this Ca2+ channel that was first shown to regulated by Ca2+ ions by Brehm and Eckert [20]. This modulation now appears to be functionally preserved in mammalian orthologs of both voltage-gated Ca2+ and Na channels, suggesting that the CI region may be a modular protein element. An explicit demonstration of such modularity is illustrated in Figure 5C. Transplantation of the CI region from NaV1.4 to CaV1.3 channels results in a chimeric channel that supports robust CDI reminiscent of wild-type NaV1.4 channels [115]. Dominant negative CaM1234 abolishes CDI observed in these chimeric channels, thus validating the successful transfer of calmodulation across the Ca2+ and Na channel superfamilies [115]. Given the common heritage of these channels dating to early eukaryotes (~ 1 billion years ago), these results argue for a persistent link between modern CI elements of Ca2+ and Na channels to a primordial Ca2+ regulatory design. It is humbling, then, to realize that CaM has likely remained a modulatory partner for these two important ion channel families for nearly all of living history.

Calmodulinopathies – a calmodulation deficiency?

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In recent years, genome sequencing of patients with long-QT syndrome (LQTS) [145–147] [148] and/or catecholaminergic polymorphic ventricular tachycardia (CPVT) [149], revealed a growing number of mutations in the three redundant genes of CaM [150] (Figure 6A). Clinically, the symptoms arising from LQTS-associated CaM mutations (LQT14/15 [151]) are resistant to conventional treatments [145–147] [148]. Could some of these disease manifestations, termed calmodulinopathies, arise from deficiencies in calmodulation of Ca2+ or Na channels? A tantalizing clue lies in prior experiments that showed how overexpression of mutant CaM with diminished Ca2+-binding capacity resulted in severe action potential prolongation in ventricular myocytes [152, 153] – a cellular correlate of LQTS. This effect was argued to result from a loss of calmodulation of CaV1.2 channels [152, 153]. Therefore, one might postulate that the naturally occurring LQTS-associated CaM mutations could perturb the same process.

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In general, many of these disease-causing mutations result in CaM molecules with substantially diminished Ca2+-binding affinity [148, 149, 154] without significant alterations to other CaM interfaces [155]. However, given the redundancy in CaM genes, it is puzzling how mutations in a single gene could account for the severity of LQT14/15. The resolution to this paradox lies in the apoCaM preassociation to certain target molecules [48]. We first consider the case of L-type Ca2+ channels [53–55]. If apoCaM binding to these channels remains intact, then the fraction of channels occupied by a mutant CaM is proportional to the total fraction of mutant CaM available in cells. This proportion of channels bound to a mutant CaM is unable to undergo CDI, thus diminishing the overall inactivation of L-type currents. In terms of the net effect on the shape of the cardiac action potential, this reduced Curr Mol Pharmacol. Author manuscript; available in PMC 2016 July 26.

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inactivation of L-type currents would result in a substantial prolongation of the action potential duration due to enhanced inward currents during the plateau and repolarization phases [27, 152, 156, 157].

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In fact, this effect of mutant CaM on L-type Ca2+ channels was recently confirmed in both recombinant and native systems [155, 158]. Firstly, live-cell FRET binding assays demonstrated no decrement in the binding affinity of mutant apoCaM to the carboxyterminus of the L-type Ca2+ channel. Secondly, the expression of LQTS mutant CaM in either HEK293 cells or ventricular myocytes resulted in a significant attenuation of CDI (Figure 6B) [155, 158]. Thirdly, in terms of cardiac cellular function, this loss of Ca2+ channel inactivation contributed to a stark and often variable prolongation of the action potential duration (APD) (Figure 6C) [147, 155]. Lastly, the increased Ca2+ influx through L-type channels also enhanced Ca2+ transient magnitudes, elevated diastolic Ca2+ concentrations, and increased Ca2+ load in the sarcoplasmic reticulum (Figure 6D) [155, 158, 159]. Importantly, these experiments also showed hints of other proarrhythmic events including electrical alternans and after-depolarizations, recapitulating the disease phenotypes reported in patients [146, 148, 149]. At the tissue level, the immense cell-to-cell functional heterogeneity introduced by mutant CaM could be a major contributor to arrhythmia in the clinical setting [160]. Together, these functional effects rationalize the severe cardiac phenotype observed in LQT14/15 patients and shed light upon novel therapeutic strategies.

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Voltage-gated Na channels also preassociate with apoCaM [81, 108, 124, 126]. As such, altered Na channel regulation may also augment the spectrum of clinical phenotypes associated with calmodulinopathies [158]. The effects of LQTS-associated CaM mutations on the cardiac NaV1.5 channel were also evaluated [158]. However, no significant alteration of the Na channel inactivation properties or the late Na current were found with the overexpression of LQTS-associated mutant CaM in either recombinant systems or ventricular myocytes[158]. These results fit with the absence of Ca2+-dependent regulation of NaV1.5 currents previously discussed [115]. Nevertheless, calmodulinopathies may alter the Ca2+-effects on NaV1.4 channels [115]. As these channels are thought to be critical for normal action potential propagation in Purkinje fibers [161], deficiencies in Ca2+ regulation may contribute to arrhythmia in patients.

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More generally, as CaM is a pervasive ion channel regulator, the pathophysiology associated with calmodulinopathies likely stems from misregulation of numerous target molecules, yet to be fully characterized. Of note, recent data suggests that CaM-ryanodine receptor interactions are also impacted by mutant CaM associated with various calmodulinopathies. These deficiencies may manifest clinically as CPVT as described elsewhere [154, 162]. The full implications of calmodulinopathies in human disease are yet to be determined; deducing further mechanistic principles and developing possible therapeutic intervention now looms on the horizon.

A shared framework of Ca2+ and Na channelopathies Given that the constituents of Ca2+ and Na channel calmodulation are similar in both genetic sequence and underlying biophysical properties, mechanisms of related disease may also


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accord to a unifying theme. A myriad of Ca2+ and Na channelopathies has been identified [163–171]. Timothy Syndrome (TS) represents one such disorder in which patients suffer from cardiac arrhythmias, immune deficiencies, and autism [172, 173]. This syndrome stems from a single de novo point mutation (either G406R or G402S) in the domain I S6 region of CaV1.2 channels [173]. The functional effects of the TS mutations on CaV1.2 is both a loss of voltage-dependent inactivation (VDI) [22, 26, 172, 173] and a reduction in CDI [26, 27]. Likewise, a similar mutation at a corresponding location of the homologous CaV1.3 channel (G407R) has been reported to be associated with autism spectral disorders [28]. Functional studies of this mutation argue for a similar slowing of Ca2+ current inactivation in both diseases [28].

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In addition, mutations in CaV1.4 that result in missense, frame-shift, and premature termination of protein translation have also been correlated with incomplete congenital stationary night blindness (CSNB), which is inherited as an X-linked trait [174, 175]. CaV1.4 regulates calcium passage into retinal photoreceptors [176–178] and serves as a major proponent in triggering glutamate release [179]. Alternative splicing of CaV1.4 results in the inclusion of an autoinhibitory domain (ICDI segment) in the distal carboxy-terminus [92]. The presence of this ICDI segment competitively displaces CaM from the channel complex thus disabling CDI [58, 75]. The heritable disorder CSNB2 has been reported to result from a truncation of this ICDI segment (K1591X), thus re-enabling CDI [58, 59]. In this manner, calmodulation of Ca2+ channels may directly contribute to retinal disorders. Likewise, aberrations in alternative splicing [90, 91] and RNA editing [95] of CaV1.3 channels in substantia nigral cells may be relevant to Ca2+ overload, suspected to be a key determinant in neurodegeneration associated with Parkinson’s disease [180–183].

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In the brain, CaV2.1 plays a critical role in determining the release of neurotransmitters at synapses in the central nervous system and may be important for synaptic plasticity [184– 186]. Furthermore, extensive genome-sequencing links genetic mutations in CaV2.1 to both familial hemiplegic migraine type 1 (FHM-1) and episodic ataxia [187, 188]. These findings provide a possible connection between FHM-1 and a mechanistic deficit for CDF [29] and channel PO [189], which may allow for possible targeted pharmacological intervention [30].

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Aberrant calmodulation of Na channels may also contribute to a wide-range of neuronal, cardiac, and muscular disorders, in accordance with the prominent role of these channels in cellular excitability and the brisk spatial propagation of action potentials [190]. The neuronal NaV1.1 channel has been heavily studied in relation to a family of epilepsy disorders, including febrile seizures [191] and Dravet syndrome [192, 193]. Interestingly, several associated channelopathic mutations have been identified on the carboxy-terminus of these channels [192, 193]. Channelopathic mutations associated with familial autism have also been identified in the related neuronal channel NaV1.2 channel [191, 194]. More specifically, an autism-associated channelopathic mutation in the CaM binding IQ domain of NaV1.2 has been argued to unveil latent calmodulation [118]. For cardiac NaV1.5 channels, various channelopathic mutations have been linked to both congenital long QT syndrome (LQTS) and Brugada syndrome [195]. The NaV1.5 associated LQTS3 is thought to result from an enhancement of late Na current [195–198], while Brugada syndrome is argued to stem from a dramatic decrement of peak Na current syndrome [195, 199, 200]. The role of


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CaM in these processes is yet to be fully elucidated. Nonetheless, many Brugada syndrome and LQTS3 associated mutations occur within the carboxyl-terminus of NaV1.5 channels [201, 202]. Lastly, for the skeletal muscle NaV1.4 channels, channelopathic mutations are associated with heritable forms of myotonia and periodic paralysis [171]. Moreover, channelopathic mutations in the carboxy-tail of NaV1.4 associated with K-aggravated myotonia [203] and cold-aggravated myotonia [204] result in a decrement of CaM regulatory function [115]. It is, thus, plausible that alterations in calmodulation may contribute to heritable forms of myotonia. The full spectrum of CaM regulatory functions and its physiological implications remain to be established across Na channel families.

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Until recently, Ca2+ and Na channels appeared to be divergent with respect to their regulatory mechanisms. An emerging theme, however, is the remarkable similarity in the conservation of CaM regulation across these channel families. This interrelation now paves the way towards a unified mechanistic framework to understand vital CaM-dependent feedback and offers shared principles to approach related channelopathic diseases.

Acknowledgments The calcium signals lab would like to thank our advisor Dr. David Yue for unwavering support, boundless kindness, and superb mentorship across the years. His recent passing has left a void in the field of ion channels and in our hearts. His enthusiasm, passion and wonder will be greatly missed. This work was supported by grants from the NHLBI (R37HL076795), NIMH (R01MH065531), NINDS (R01NS073874) and NINDS (R01NS08074)to DTY; AHA(13PRE16500029) to WL, NSF(DGE-1232825) to JN and MSTP fellowships to JBI and SRL.

Trainees of the Calcium Signals Laboratory Author Manuscript

Manu Ben-Johny*, Ivy E. Dick*, Wanjun Yang*, John B. Issa*, Lingjie Sang*, Shin Rong Lee*, Worawan B. Limpitikul*, Jacqueline Niu*, Po Wei Kang*, Rahul Banerjee*, Jennifer S. Babich*, Ho Namkung*, Jiangyu Li*, Manning Zhang*, Philemon S. Yang*, Daniel N. Yue*. *We are the calcium signals lab, and we are proud to have been trained by David Yue.

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203. Kubota T, Kinoshita M, Sasaki R, Aoike F, Takahashi MP, Sakoda S, Hirose K. New mutation of the Na channel in the severe form of potassium-aggravated myotonia. Muscle Nerve. 2009; 39:666–673. [PubMed: 19347921] 204. Wu FF, Gordon E, Hoffman EP, Cannon SC. A C-terminal skeletal muscle sodium channel mutation associated with myotonia disrupts fast inactivation. J. Physiol. 2005; 565:371–380. [PubMed: 15774523]

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Figure (1).

A) Convenient experimental setup to examine CDI of Ca2+ channels. Left, no feedback regulation triggered upon Ba2+ influx through channel. Right, Ca2+ influx triggers Ca2+ regulation. B) Typical Ca2+ and Ba2+ current profiles through CaV1.3 channel. In response to a step depolarization, the Ca2+ current (red trace) activates and then sharply inactivates. Ba2+ current (black trace), does not inactivate. Adapted with permission [115]. C) Diagram depicting regions of Ca2+ channels critical for calmodulation, including dual vestigial EF hands (pink and green), IQ region (blue) on the C-terminus and NSCaTE (black) on the N-


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Author Manuscript

terminus of the channel. D) Mechanist scheme for CaM regulation of Ca2+ channels. In the E state, the channel is devoid of CaM and incapable of undergoing CDI. Following apoCaM binding (A state), the channel can undergo N- or C-lobe-driven CDI, state IN and IC, respectively. Configuration ICN corresponds to a fully inactivated state where N-and C-lobe of CaM engages respective effector interfaces. E) Molecular model of apoCaM preassociation to CaV1.3. The N-lobe of apoCaM preassociates with the dual vestigial EF hand segments, and the C-lobe with the channel IQ domain. F) Proposed model of Ca2+/CaM interaction with channel interfaces. Upon Ca2+ binding, the N-lobe of Ca2+/CaM interacts with the NSCaTE segment. The C-lobe of Ca2+/CaM forms a tripartite complex with the channel dual vestigial EF hands and the IQ domain. Adapted with permission [41].

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Author Manuscript Author Manuscript Figure (2).

Author Manuscript

A) Functional bipartition of CaM in CaV2.1 channel. Top, CaV2.1 currents evoked by a train of action potentials. Rapid Ca2+ dependent facilation of Ca2+ current (circle, bottom subpanel) is followed by Ca2+-dependent inactivation (square, bottom subpanel). Adapted with permission [70]. B) Distinct modes of spatial Ca2+ signaling. Under high Ca2+ buffering, Ca2+ elevations are restricted to the channel nanodomain (left). Low Ca2+ buffering permits global elevation of cytoplasmic Ca2+ (right). The resident CaM molecule can decode these distinct Ca2+ signals Adapted with permission [83]. C) Table summarizes dual regulation of CaV channels by the two lobes of CaM. Adapted with permission [16].

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Figure (3).

A) Milestones in Na channel (left) and Ca2+ channel (right) calmodulation. B) Experimental scheme to study Ca2+ regulation of Na channels. Na current properties are evaluated from two population of cells dialyzed internal solutions containing either low and high Ca2+ concentrations C). Protocol used to determine steady-state inactivation of Na channels (left). Expected Ca2+ dependent effects for two well-studied Na channel families. Adapted with permission [115].


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Author Manuscript Author Manuscript Author Manuscript

Figure (4).

A) Na channel regulation probed using rapid delivery of Ca2+ by photouncaging. Schematic shows simultaneous patch-fluorimetry and Ca2+ photouncaging. Ca2+ measured quantitatively using ratiometric fluorescence measurement. Ca2+ elevations are triggered using a brief UV pulse. B) Absence of Ca2+ regulation of cardiac Na channels (NaV1.5) in a heterologous (HEK293) system. C) Ca2+ regulation of native NaV1.5 current from ventricular myocytes. D) Robust Ca2+-dependent inactivation in heterologously expressed NaV1.4 channels. Here, intracellular Ca2+ elevation decreases peak Na current (second row). Population data shows a dose-dependent response of CDI to intracellular Ca2+ (bottom). E) Robust Ca2+-dependent inactivation of native NaV1.4 currents from skeletal myotubes (GLT cells). Adapted with permission [115].


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Figure (5).

A) Table shows conservation of Ca2+/CaM regulation of Ca2+ and Na channels. B) Phylogenetic tree of the Ca2+ and Na channel superfamilies. HVA, high-voltage activated Ca2+ channels. LVA, low-voltage activated Ca2+ channels. C) Modularity of CI region. Transplanting CI region of NaV1.4 to CaV1.3 supports CDI. Coexpression of mutant CaM1234 abolishes Ca2+ regulation of these chimeric channels. Adapted with permission [115].

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Fig. (6).

Author Manuscript

A) Sequence alignment of the two lobes of CaM. The EF hand segments are shaded rose and green. LQTS-associated CaM mutations are highlighted. B) Reduction of CDI of Ca2+ currents in adult guinea pig ventricular myocytes (aGPVMs) expressing CaMD96V (red), compared to CaMWT (black). Ba2+ current through the same channels (gray) shows the extent of VDI in these cells. C) Averaged action potentials from aGPVMs expressing CaMWT (black) and CaMD96V (red). Shaded regions represent standard deviation. CaMD96V significantly prolongs the APD. D) Averaged Ca2+ transient waveforms from aGPVMs expressing CaMWT (black) and CaMD96V (red). Shaded regions represent standard deviation. CaMD96V significantly increases the Ca2+ transient magnitude. Adapted with permission [155].


Calmodulin regulation (calmodulation) of voltage-gated calcium channels.

Towards a unified theory of health-disease: II. Holopathogenesis.

A unified theory of calcium alternans in ventricular myocytes.

calmodulin regulation across Na and Ca2+ channels.

Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.

Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

Calmodulin is responsible for Ca2+-dependent regulation of TRPA1 Channels.

Regulation of mTORC1 by lysosomal calcium and calmodulin.

Unified theory of anesthesia.

A new approach to homeostatic regulation: towards a unified view of physiological and ecological concepts.

SK channels and calmodulin.

A 'unified theory' of prion propagation.

Towards a unified theory for immunogenetic systems. IV. A complex-simple probe into the Ag system.

Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels.

Calmodulin methylation: another layer of regulation in calcium signaling.

Regulation of Lung Epithelial Sodium Channels by Cytokines and Chemokines.

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1.

Modulation of L-type calcium channels by sodium ions.

Homologous and heterologous regulation of voltage-dependent calcium channels.

Calcium and calmodulin.

Toward a unified theory of voice production and perception.

Towards a Unified Mental Health Service.

Differential Regulation of PI(4,5)P2 Sensitivity of Kv7.2 and Kv7.3 Channels by Calmodulin.

Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency.